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| Targets |
Marker dye
DNA minor groove, specifically AT-rich regions of double-stranded DNA. meta-iodoHoechst 33258 is a marker dye from the Hoechst series that binds nucleic acids by binding to the minor groove in the DNA double strand. Hoechst dyes preferentially bind to AT-rich DNA sequences. The introduction of an iodine atom into the Hoechst 33258 structure enhances its chemical properties and biological applications. |
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| ln Vitro |
Preparation of Hoechst working slution:
1.1 Preparation of stock solution Prepare 1 mg/mL Hoechst stock solution with DMSO. Note: Hoechst stock solution is recommended to be aliquoted and stored in the dark at -4 ℃ or -20 ℃. 1.2 Preparation of working slution Dilute the stock solution with preheated serum-free cell culture medium or PBS to a final concentration of 10 μ g/mL Hoechst working solution. Note: Please adjust the concentration of Hoechst working solution according to your specific needs, and use freshly prepared solutions. 2. Cell staining (suspended cells) 2.1 Centrifuge and collect cells, wash twice with PBS for 5 minutes each time. Cell density is 1 × 10~6/mL 2.2 Add 1 mL of Hoechst working solution and incubate at room temperature for 3-10 minutes. 2.3 400 g, centrifuge for 3-4 minutes, discard the supernatant. 2.4 Wash the cells twice with PBS, each time for 5 minutes. After resuspending cells in 1 mL serum-free medium or PBS, observe them using a fluorescence microscope or flow cytometer. 3. Cell staining (adherent cells) 3.1 Cultivate adherent cells on sterile coverslips. 3.2 Remove the cover glass from the culture medium and aspirate excess culture medium. 3.3 Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 3-10 minutes. 3.4 Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and observe using a fluorescence microscope or flow cytometer. In vitro, meta-iodoHoechst 33258 is used as a fluorescent DNA stain for DNA quantitation and fluorescence microscopy. The dye exhibits blue/cyan fluorescence upon binding to DNA and can be visualized using UV excitation. It is used in flow cytometry, fluorescence microscopy, and other applications requiring DNA staining. The meta-isomer offers enhanced brightness and photostability compared to the parent compound. It has been studied for its DNA breakage and photosensitization properties. |
| ln Vivo |
In vivo studies of meta-iodoHoechst 33258 are limited as the compound is primarily used as a research tool for in vitro applications. The compound is not intended for therapeutic use in animals or humans. Its in vivo effects would be related to its DNA-binding properties. Further in vivo studies are needed to fully characterize its biological effects.
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| Enzyme Assay |
DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions[1].
In vitro DNA binding assays for meta-iodoHoechst 33258 measure its affinity for DNA. The assay uses purified double-stranded DNA (e.g., calf thymus DNA or synthetic AT-rich oligonucleotides) and measures the fluorescence enhancement upon dye binding. Varying concentrations of the dye are incubated with DNA in buffer, and fluorescence intensity is measured at appropriate excitation/emission wavelengths (Ex/Em ~350/461 nm). The binding affinity (Kd) is calculated from titration curves. Competition assays with unlabeled Hoechst 33258 can be used to assess relative binding affinities. |
| Cell Assay |
In vitro cell-based assays for meta-iodoHoechst 33258 use cultured cells for DNA staining and quantitation. Cells are fixed and permeabilized, then incubated with the dye at concentrations of 0.1-10 microg/mL for 15-30 minutes. After washing, DNA content is measured by flow cytometry or fluorescence microscopy. For photosensitization studies, cells are treated with the dye and exposed to UVA irradiation. DNA damage is assessed by gammaH2AX staining or comet assays.
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| Animal Protocol |
In vivo animal studies for meta-iodoHoechst 33258 are not typically performed as the compound is used as a research probe rather than a therapeutic agent. If performed, typical protocols would involve administration of the compound via intravenous or intraperitoneal injection. Tissue samples would be collected for fluorescence imaging or DNA content analysis. However, such studies are not extensively reported in the literature.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of meta-iodoHoechst 33258 are characteristic of small-molecule fluorescent dyes. With a molecular weight of 534.39 and a molecular formula of C25H23IN6, the compound is soluble in DMSO. It is typically stored as a powder at -20degC. As a research dye, pharmacokinetic studies are not typically performed.
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| Toxicity/Toxicokinetics |
meta-iodoHoechst 33258 is intended for research use only and is not for human therapeutic use. Standard safety precautions for handling chemical compounds apply. The compound is not approved for clinical use.
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| References | |
| Additional Infomation |
This article describes a variety of applications of fluorescence microscopy for detecting deoxyribonucleic acid synthesis. These applications include: (a) analyzing the exchange and separation of sister chromatids during mitosis; (b) locating deoxyribonucleic acid regions on chromosomes containing thymine residues asymmetrically distributed among polynucleotide chains; and (c) detecting late replication regions in metaphase chromosomes. In both fixed cytological specimens and unfixed cultured cells, it was confirmed that the biosynthesis of 5-bromodeoxyuridine incorporated into the interphase nucleus could inhibit Hoechst 33258 fluorescence. Many of the cytological observations described in this article may lay the foundation for future biochemical research. [2]
meta-iodoHoechst 33258 is a research-grade iodinated DNA-binding bibenzimidazole and fluorescent DNA stain. It is a mono-iodinated derivative of Hoechst 33258 with a molecular formula of C25H23IN6 and a molecular weight of 534.39. The compound is excited by UV light at around 350 nm and emits blue/cyan fluorescence at 461 nm. It is used in flow cytometry, fluorescence microscopy, and photosensitization studies. Not approved for clinical use. |
| Molecular Formula |
C25H23IN6
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|---|---|
| Molecular Weight |
534.394796609879
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| Exact Mass |
534.103
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| CAS # |
158013-42-4
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| PubChem CID |
449492
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| Appearance |
Off-white to pink solid powder
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| LogP |
5.132
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
32
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| Complexity |
642
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
ZEXOKHKEQNNOBG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H23IN6/c1-31-9-11-32(12-10-31)19-6-8-21-23(15-19)30-25(28-21)17-5-7-20-22(14-17)29-24(27-20)16-3-2-4-18(26)13-16/h2-8,13-15H,9-12H2,1H3,(H,27,29)(H,28,30)
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| Chemical Name |
2-(3-iodophenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole
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| Synonyms |
meta-iodoHoechst 33258; 158013-42-4; 2-(3-iodophenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole; Metaiodohoechst; CS-1310; meta-iodoHoechst33258; SCHEMBL26252728;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :< 1 mg/mL
Ethanol :< 1 mg/mL |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8713 mL | 9.3565 mL | 18.7129 mL | |
| 5 mM | 0.3743 mL | 1.8713 mL | 3.7426 mL | |
| 10 mM | 0.1871 mL | 0.9356 mL | 1.8713 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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